The vast majority (>85%) of human cancers use telomerase as the primary telomere length maintenance mechanism. My long-term goal is to establish an independent research laboratory, where I will elucidate the molecular mechanisms by which alternative splicing plays a role in telomere length maintenance mechanisms. This basic biological information will be important for my long-term goals for designing telomerase specific cancer therapeutics. The K99/R00 career award will help me in achieving my goals as a research scientist by advancing my training in: gene expression regulation, alternative splicing, next generation sequencing, and molecular biology under the co-mentorship of Dr. Woodring Wright, as well as cancer cell biology and models of cancer cell phenotypes under the co-mentorship of Dr. Jerry Shay. This training and guidance will compliment my previous training in gene expression, muscle biology, and cell signaling. The scientific environment at UT Southwestern Medical Center is extremely stimulating and will provide me with the facilities necessary for the completion of the mentored phase of this project and will prepare me to transition smoothly into the independent faculty phase. hTERT expression is developmentally regulated and has several splice variants. Only full-length hTERT mRNA produces catalytically active telomerase. Upon differentiation into mature tissue telomerase activity is down-regulated and only non-catalytically active hTERT splice variants remain. However, upon transformation to cancer, the majority of cancer types express full-length hTERT and have functional telomerase activity that helps to maintain the already very short telomeres in precancerous lesions. The transcriptional activation of hTERT has been widely studied, but the regulation of hTERT alternative splicing is almost entirely unknown. In Aim 1, during the K99 phase, I will define the role of NOVA1 that I identified in two different RNAi screens, in the regulation of hTERT splicing, telomere length, and telomerase activity. I will determine if NOVA1 binds hTERT pre-mRNA directly (CLIP) or in a protein complex (RIP). I will also define the role of NOVA1 in cancer cell alternative splicing by RNA sequencing. In Aim 2, during the K99 phase, I will use two xenograft models of tumor progression to determine if reduced levels of NOVA1 result in less metastases and tumor progression via inhibition of telomerase. During the R00 phase, Aim 3, I will characterize the regulation of NOVA1's 3'UTR in cancer cells and the potential of using a NOVA1 3'UTR reporter for small molecule screening to find a novel telomerase inhibitor. I will also continue to decipher the hTERT splicing code I discovered during my mentored phase with the goal of finding novel means to inhibit telomerase in cancer. These studies should lead to a robust research program into how alternative spicing participates and regulates telomere length maintenance mechanisms in cancer cells.